1. Field of the Invention
The invention relates generally to compositions and methods for facilitating the construction of recombinant nucleic acid molecules, and more specifically to compositions useful for covalently linking two or more nucleic acid molecules, including for directionally or non-directionally linking the nucleic acid molecules, and to methods of generating such covalently linked recombinant nucleic acid molecules.
2. Background Information
The ability to clone large numbers of nucleotides sequences, including gene sequences and open reading frames allows a great deal of information to be obtained about gene expression and the regulation thereof. In addition, such sequences can be useful for understanding the etiology of disease conditions and, ideally, can provide a means to diagnose and treat such diseases. However, while it is relatively simple matter to clone large numbers of expressed nucleotides sequences, for example, it is a more difficult undertaking to characterize the regulatory elements involved in the expression of such sequence and to properly express a polypeptide encoded by the sequence. In particular, there is a need for improved methods for ligating nucleic acid molecules and cloning nucleic acid molecules such that a functional recombinant nucleic acid molecule is produced. There is a particular need for directional cloning methods, wherein an insert can be cloned into a vector or linked to one or more other nucleic acid molecules in a predetermined orientation.
The use of topoisomerases provides a convenient means to improve cloning and ligation methods. For example, the use of topoisomerase to perform rapid ligation of polymerase chain reaction (PCR) products into a vector has cut traditionally laborious cloning methods down to a five minute procedure. As such, topoisomerase is particularly useful for high throughput cloning applications. However, given the current demand for expressing open reading frames (ORF) in genome scale molecular cloning procedures, there still remains a need to better control the orientation in which two or more nucleic acid molecules are linked such that functional recombinant nucleic acid molecules such as expressible cloned nucleic acid molecules can be prepared.
Expression of cloned ORFs demands that the PCR product be inserted into the vector in its correct orientation, so as to work in accord with functional expression domains located on the vector. In the current state of the art for topoisomerase mediated cloning, ORFs are amplified by PCR using various DNA polymerases. A polymerase such at Taq, which does not have a proof-reading function and has an inherent terminal transferase activity, is commonly used, and produces PCR products containing a single, non-template derived 3′ A overhang at each end. These amplification products can be efficiently cloned into topoisomerase-modified vectors containing a single 3′ T overhang at each end (TOPO TA Cloning® Kit, Invitrogen Corp., Carlsbad, Calif.). In comparison, a polymerase such as pfu, which has an inherent 3′ to 5′ exonuclease proof-reading activity, produces PCR products that are blunt-ended. Topoisomerase-modified vectors containing blunt ends are available for cloning of PCR products produced with proofreading polymerases (Zero Blunt TOPO® PCR Cloning Kit, Invitrogen Corp., Carlsbad, Calif.). Incubation of either PCR product and the proper topoisomerase-modified vector results in five minute ligation. However, the orientation of the insert obtained using such cloning methods is random.
Because the orientation of DNA fragment insertion into topoisomerase-modified cloning vectors is random, users must screen clones to identify those having the proper orientation. Insert orientation can be determined using various methods including, for example, restriction enzyme analysis, in vitro transcription from vector-encoded promoter elements, and PCR using, for example, one insert-specific primer and one vector-specific primer. As is evident, however, the requirement for determining insert orientation requires an investment of time and can substantially increase the cost for identifying a nucleic acid molecule of interest, particularly where a high throughput cloning method is used. As such, current cloning methods are severely limited, particularly for high throughput gene expression analysis for several reasons, because numerous laborious steps must be performed in order to select clones with correctly oriented inserts, and there is a need to screen as many as eight colonies of each clone to identify one having the proper orientation. Thus, a need exists for methods and reagents that are useful for covalently linking two or more nucleic acid molecules in a directional orientation. The present invention satisfies this need and provides additional advantages.